Propagation of commercially important tree species for reforestation of managed forests has assumed great importance as the urban expansion and the demand for wood increases while world forests continue to disappear at an alarming rate. Tree nurseries now supply seedlings in the tens of millions annually. Most of these seedlings are grown from seed produced in seed orchards stocked with genetically selected trees. However, full genetic gain is usually not realized since most orchards are open pollinated; i.e. by wild pollen of unknown origin and genetic quality. A very small amount of full sib seed is produced in which both cone and pollen parents are controlled. However, this seed is very expensive since its production is extremely labor intensive. Immature female strobili must be covered with bags to prevent entry of wild pollen. Selected pollen is then injected into the bags at the time when the female strobili are most receptive to fertilization. Little of this full sib seed ever finds its way directly into the forest as nursery seedlings because of the high cost. Much is used in nurseries as stock for producing rooted cuttings. Others is dedicated for progeny trials for selection of stock for future generation seed orchards.
Tissue culture is one method by which plants of known genetic characteristics have been produced for many years. Originally this procedure was carried out only on ornamental species. Various orchid varieties and Boston fern might be cited as examples. Extensive research on conifers came later, in part because they were far more intractable. The earliest successful procedures with conifers were based on organogenesis. Portions of a newly sprouted seedling were placed on a bud-inducing medium. The buds were then separated and placed on other media for further propagation and root development. Two problems prevented wide use. The process was highly labor intensive and genetic mutation was common.
Clonal propagation of forest species by embryogenesis appeared to be a more promising route, although the technical obstacles have been severe. The first successful embryogenesis of a conifer was not achieved until about 1975. For the decade thereafter the number of tissue culture propagated embryos that were successfully converted into growing trees was probably no more than a few dozen. A large body of related literature now exists including a significant number of patents. As exemplary United States patents the following might be cited: Gupta et al. U.S. Pat. No. 5,036,007; Roberts U.S. Pat. No. 5,183,757; Uddin U.S. Pat. No. 5,187,092; Pullman et al. U.S. Pat. No. 5,294, 549; Becwar et al. U.S. Pat. No. 5,413,930 Attree et al. U.S. Pat. No. 5,464,769; and Gupta U.S. Pat. No. 5,563,061. These patents deal primarily with the propagation of Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco), loblolly pine (Pinus taeda L.) and various spruce (Picea) species. The procedures share many features in common. An explant, often an immature seed or the embryo from an immature seed, is placed on a gelled initiation medium. This medium usually will contain plant growth hormones from the groups known as auxins and cytokinins. If initiation is successful a gelatinous mass containing multiple immature embryos will be generated in several weeks. This mass is then removed and subcultured on a maintenance and multiplication medium which may be gelled, liquid, or some combination of these. The maintenance medium will typically have a reduced concentration of plant growth hormones. Subcultures to fresh medium arc required on a regular basis. While the clone is on the maintenance medium, embryo size growth is limited. Embryos from maintenance may then be placed on a development medium. This normally lacks the auxins and cytokinins but may instead include the hormone abscisic acid. Here somatic embryos develop into cotyledonary embryos with a size and morphology that closely resembles their mature zygotic counterparts. A further treatment following development may be a partial desiccation after which the embryos are placed on a hormone free germination medium. During the next several weeks radicle and epicotyl elongation will occur. The germinants or emblings are then removed and placed in soil for further development into plantlets. After a period of greenhouse growth they may then be outplanted. Alternatively, the embryos removed from the development medium may be placed in manufactured seeds; e.g., as shown in Carlson et al. U.S. Pat. No. 5,236,469.
Even though great progress has been made in conifer tissue culture in the past two decades it is only now beginning to be of commercial significance as a source of reforestation stock. For some species and for particular clones of any species, propagation by tissue culture has been far less successful than desired. Significant biological and engineering problems remain. One problem has been that conversion percentage from somatic embryos to plants growing in soil has frequently been lower than desired. Often as low as 10% of the transplanted germinants will survive and 40% survival has been considered good. It is interesting that this survival percentage also holds for excised zygotic embryos. A further problem is logistical rather than biological. Unless the conifer somatic embryos are to be processed into manufactured seeds, they are presently individually removed from the sterile development medium by hand and placed on a germination medium. After an appropriate time those embryos that germinate are placed in a potting soil mixture for further growth. While this poses no problem on a laboratory scale it does for automation on a production basis when many hundreds of thousands of plants may be wanted. A method has been needed to accumulate germinants that may have become available over a period of days or weeks so that potting might be done within a relatively short time interval.
It is known that certain vegetable seeds may be pregerminated and stored in aerated water or at high humidity at temperatures of 0.degree. C. to 4.degree. C. for several days and then planted without loss of viability. As examples see Finch-Savage, Annals of Applied Biology 97: 345-352 (1981) or Wurr et al., pp 335-343 of the same journal. Similar treatment has also been used on ornamentals such as snapdragon; e.g., Frazier et al., Journal of the American Society of Horticultural Science, 107 (4): 660-664 (1982). In all of these cases pregermination was apparently allowed to proceed only to the point of radicle emergence prior to the wet cold storage treatment. Gerbera daisy embryos produced by tissue culture and rooted have been stored cold on filter paper in closed boxes. Moisture conditions were not clearly specified; e.g., Hempel et al., Rosliny Ozdobne Series B, 10: 85-90 (1985). To the present applicant's knowledge similar techniques have not been tried with conifer somatic embryo germinants or on any plants in which a well developed epicotyl has already been formed.
The present invention addresses the problem of accumulation and storage of any desired quantity of conifer somatic embryo germinants without loss of vigor until they can be conveniently and efficiently planted. The method further significantly improves longer term conversion success from germinants to plants ready to set out in the forest.